Deactivation, catalyst enzyme

Compared to synthetic catalysts, enzymes have many advantages. First of all, being natural products, they are environmentally benign and therefore their use does not meet pubhc opposition. Enzymes act at atmospheric pressure, ambient temperature, and at pH between 4 and 9, thus avoiding extreme conditions, which might result in undesired side reactions. Enzymes are extremely selective (see below). There are also, of course, some drawbacks of biocatalysts. For example, enzymes are known in only one enantiomeric form, as they consist of natural enantiomeric (homochiral) amino acids their possible modifications are difficult to achieve (see Section 5.3.2) they are prone to deactivation owing to inappropriate operation parameters and to inhibition phenomena. 

Ionic liquids are generally regarded as highly stable, and the widely used dial-kylimidazolium ionic liquids are indeed thermostable up to 300 °C [4]. The propensity of the [BF4] and [PF6] anions to hydrolyze with liberation of HF [37], which deactivates many enzymes, has already been mentioned. The [TfO] and [ Tf2N] anions, in contrast, are hydrolytically stable. Dialkylimidazolium cations have a tendency to deprotonate at C-2, with ylide (heterocarbene) formation. Such ylides are strong nucleophiles and have been used as transesterification catalysts, for example [38]. These could cause enzyme deactivation as well as background transesterification when formed in small amounts from anhydrous ionic liquids and basic buffer salts, for example. 

Removing the product as fast as possible from the reaction mixture could possibly prevent the enzyme from deactivating. This was experimentally tried on pilot scale using an ultrafiltration unit, parallel to the reactor. In this filtration unit the product is separated and the unreacted components and the enzyme are returned to the reactor. An even better option, using a true membrane reactor in which the catalyst (enzyme) would remain on one side and the product would remain on the other side, was not tested. Both options are given schematically in Figure 7. 

However, to our knowledge, most previous studies of enzyme-catalyzed polymerizations have avoided temperatures > 90 oC, which is likely due to thermal deactivation of enzyme catalyst (13-15). It has been found that enzyme immobilization can improve the stability and recyclablity of native enzyme (16). Silica particles, activated by methanesulfonic acid, are effective and economic inorganic carriers for enzyme immobilization (17). Herein, we present a minireview of our works about immobilized porcine pancareas lipase on silica particles (IPPL) for polymer synthesis, such as polycarbonates, polyesters, polyphosphates and their copoljmiers. 

Catalysts are often specific, i.e. they will only afiect the rate of a particular reaction. This is particularly true of some biological catalysts (enzymes). Enzymes are proteins. They are often deactivated (or even destroyed) if the temperature rises much above 40 °C (Fig. 14.9). Most enzymes operate in a narrowband of pH, and many require the presence of a metal ion or another complex organic molecule as cofactor. For more about enzymes, see Case Study 1 on the website. 

Lee and Reilly (1981) defined a more rigorous form of the Thiele modulus based on the generalized modulus of Bischolf and Aris (see Chapter 7) which is particularly useful in analyzing the role of diffusion in deactivation. Their analysis shows that, in reactant-independent deactivation, the presence of a strong dilfusional limitation lowers the rate of deactivation to half the diffusion-free value. Thus, surprisingly, diffusion seems to have a favorable effect on the performance of a deactivating immobilized enzyme catalyst. 

The comparison of TOFs and TONs of different (bio)catalysts should be exercised with great caution, since these numbers only indicate how fast the catalysts act at the onset of the reaction within a limited time span, but they do not tell whether the activity remains at a constant level or if it dropped due to catalyst/ enzyme deactivation. 

Enzymes are important catalysts in biological organisms and are of increasing use in detergents and sensors. It is of interest to understand not only their adsorption characteristics but also their catalytic activity on the surface. The interplay between adsorption and deactivation has been clearly illustrated [119] as has the ability of a protein to cleave a surface-bound substrate [120]. 

This is the first example of a reaction for which the presence of a chelating ligand was observed to facilitate rather than retard metal-catalysed epoxidation (Gao et al., 1987). It was found that the use of molecular sieves greatly improves this process by removing minute amounts of water present in the reaction medium. Water was found to deactivate the catalyst. All these developments led to an improved catalytic version that allows a five-fold increased substrate concentration relative to the stoichiometric method. Sensitive water-soluble, optically active glycidols can be prepared in an efficient manner by an in situ derivatisation. This epoxidation method appears to be competitive with enzyme-catalysed processes and was applied in 1981 for the commercial production of the gypsy moth pheromone, (-1-) disparlure, used for insect control (Eqn. (25)). 

Unlike in the previous example, here the catalysts are not reported specifically to deactivate one another. Rather, the immobilization of the lipase in these silica elastomer spheres allows access to reaction conditions that are otherwise unavailable, namely higher temperatures, as the lipase is no longer deactivated and is able to undergo multiple reaction cycles, resulting in a much higher enzyme productivity, in terms of mass of product per unit mass of protein. The activity of the lipase is also observed to have increased, possibly not only from its ability to access higher temperatures when immobilized, but also due to the increased stability of the active... 

When supported complexes are the catalysts, two types of ionic solid were used zeolites and clays. The structures of these solids (microporous and lamellar respectively) help to improve the stability of the complex catalyst under the reaction conditions by preventing the catalytic species from undergoing dimerization or aggregation, both phenomena which are known to be deactivating. In some cases, the pore walls can tune the selectivity of the reaction by steric effects. The strong similarities of zeolites with the protein portion of natural enzymes was emphasized by Herron.20 The protein protects the active site from side reactions, sieves the substrate molecules, and provides a stereochemically demanding void. Metal complexes have been encapsulated in zeolites, successfully mimicking metalloenzymes for oxidation reactions. Two methods of synthesis of such encapsulated/intercalated complexes have been tested, as follows. 

In this context the lipase was immobilized on a support which also adsorbed water and propionic acid. During the reaction, the water caused a decrease of the reaction rate. While the water adsorption on the catalyst results in a reversible decrease of the enzyme activity, an excessive accumulation of water in the bulk mobile phase resulted in rapid irreversible deactivation of the enzyme. 

There are at least three reasons for attempting to prepare solid-phase catalysts that resemble enzymes. Synthetic procedures would generally be simplified. Catalytic groups are fixed on the support so that they cannot interact with one another, for example, thiols cannot deactivate by forming disulfides and metal ions cannot deactivate by forming binuclear structures. Finally, if the successful catalyst is eventually made, it will almost certainly be used in heterogeneous systems. 

Catalysts and enzymes also can vary significantly between batches and exhibit activation and deactivation, so that reaction rates may be expected to vary with time. Thus it is not unusual to find that a reaction activation energy increases with the time that a process has been onstream, which one might need to fit by assuming that E is a function of time. As you might expect problems such as these require careful consideration and caution. We win consider catalytic reactions and their kinetics in Chapter 7. 

Imbalance in the stoichiometry of polycondensation reactions of AA-BB-type monomers can be overcome by changing to heterofunctional AB-type monomers. Indeed, IIMU has been subjected to bulk polycondensation using lipases as catalyst in the presence of 4 A molecular sieves. At 70 °C, CALB showed 84% monomer conversion and a low molecular weight polymer (Mn 1.1 kDa, PDI 1.9). No significant polymerization was observed with other lipases (except R cepacia lipase, 47% conversion, oligomers only) and in reference reactions with thermally deactivated CALB or in the absence of enzyme. Further optimization of the reaction conditions (60wt% CALB, II0°C, 3 days, 4 A molecular sieves) gave a polymer with Mn of 14.8 kDa (PDI 2.3) in 86% yield after precipitation [42]. 

The catalysts were found to be stable up to 323 K without any deactivation. An activation energy of 45 kJ/mol was found for the free enzyme, 41 kJ/mol for the immobilized lipase, indeed confirming the absence of mass transfer limitations. 

Protein catalyst stability is limited. This is one of major drawbacks of enzymes. They commonly require temperatures around ambient to perform (15-50°C), pH values around neutral (pH 5-9), and aqueous media. In addition, any number of system components or features such as salts, inhibitors, liquid-gas or liquid-solid interfaces, or mechanical stress can slow down or deactivate enzymes. Under almost any condition, native proteins, with their Gibbs free enthalpy of stability of just a few kilojoules per mole, are never far away from instability. In this book, we cover inhibitors (Chapter 5, Section 5.3) or impeding system parameters (Chapter 17) and successful attempts at broadening the choice of solvents (Chapter 12). 

High enzyme concentration. The reaction rate and s.t.y. can be enhanced by increasing the catalyst concentration [E] in practice, however, in contrast to the formalism of Eq. (2.3), owing to an either excessive viscosity increase or excess of deactivated protein in the reactor, a maximum limit of enzyme concentration is reached. 

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